A two-phase study model for the standardization of HER2 immunohistochemical assay on invasive ductal carcinoma of the breast.

OBJECTIVES: To develop and verify a standardized protocol for HER2 immunohistochemical assays on invasive ductal carcinoma of the breast in Thailand. MATERIAL AND METHOD: A two-phase study approach was employed. In the Phase One, after verifying the proposed protocol that adopted the HercepTest procedure using readily available primary antibodies, CB11 and A0485, Lab 1 performed the HER2 immunohistochemical staining for 137 cases of invasive ductal carcinoma twice with two types of the antibody. Nine pathologists from 8 centers independently examined and scored all the 2 x 137 stained slides that were blinded for antibody type. Interobserver reliability was calculated using pair-wise kappa. Following discussion of the results, the Phase Two study was planned. Lab 2 and Lab 3 independently performed the HER2 staining according to the protocol for 60 invasive breast carcinoma cases. The same group of pathologists scored 2 x 60 stained slides that were masked for laboratories. Interobserver reliability and interlaboratory agreement from each pathologist were calculated using kappa statistics. Three interpreted categories--namely negative, equivocal and positive tests were used in the analyses. RESULTS: Phase One study showed interobserver agreement between pairs varied from kappa 0.75 (95%CI, 0.68-0.82) to 0.06 (95%CI, 0-0.14) while Phase Two study obtained pair-wise kappa scores ranged from 0.84 (95%CI, 0. 80-0.89) to 0. 65 (95%CI, 0.59-0.71). Interlaboratory kappa for each pathologist was 0.67 (95%CI, 0.61-0.73). CONCLUSION: The standardization of HER2 immunohistochemical assay was achieved through this two-phase study model. It had added benefits of improving pathologists' expertise and verifying the HER2 testing protocol to be used in Thailand.

Efficacy of immunotherapy using antigens of Pythium insidiosum in the treatment of vascular pythiosis in humans.

Human pythiosis is an emerging disease in the tropical, subtropical and temperate regions of the world. It is caused by the straminipilan, fungus-like, aquatic organism Pythium insidiosum. Pythiosis occurs in localized as well as systemic or vascular forms. Most patients with arterial pythiosis usually have underlying hematologic disorders such as thalassemia and aplastic anemia/paroxysmal nocturnal hemoglobinuria (PNH) syndrome. Vascular pythiosis is characterized by ascending blood vessel infections and thrombosis of the major arteries especially those of the lower extremities. When the infection reaches a main artery, the patient usually dies within weeks. Since this pathogen is resistant to most antifungal drugs, immunotherapy was recently used to cure humans and animals with the disease. A modified P. insidiosum-antigen (PIA) formulation had already saved a young boy with life-threatening arterial pythiosis. Here, we report the therapeutic benefits of the PIA in eight patients with vascular pythiosis. Six of them had thalassemia and the other two had PNH. All of the patients had arterial occlusion of the lower limbs. P. insidiosum was isolated and identified by culture and by histopathology. All patients had evidence of active infection when immunotherapy began. After two injections of 100-200 microl of PIA (2.0mg/ml), at a 14-day interval, four patients (50%) had dramatic and complete remission. Two patients showed partial responses to PIA while the other two did not. Clinical responses correlated with the immunological reactions at the site of injection, clearance of the arteries and cytokine production. The latter included the shifting in serum levels of IL4 and IL5 to IL2 suggesting a switching from a T helper 2 (Th2) to a T helper 1 (Th1) subset. Our findings provide further evidence that immunotherapy using PIA is a safe and effective method to treat pythiosis in humans.